Diagnostics and therapy of diseases associated with HHV-8 infections

ABSTRACT

The present invention is directed to monoclonal antibodies capable of specifically binding to and recognizing an antigenic determinant (epitope) of the protein kaposin or a derivative therof, hybridoma cell lines producing said monoclonal antibodies, diagnostic systems for the detection of the presence of a kaposin protein or a derivative thereof as well as antibodies directed against the kaposin protein or a derivative thereof, methods for detection of the expression of kaposin protein or a derivative thereof in a biological sample, methods for the detection of antibodies directed against kaposin protein or a derivative thereof, uses of the monoclonal antibodies provided according to the invention and uses of the kaposin protein or a derivative thereof, each in diagnostics and therapy.

The present invention is directed to monoclonal antibodies capable ofspecifically recognizing an antigenic determinant (epitope) of theprotein kaposin or a derivative therof and capable of binding thereto,hybridoma cell lines producing said monoclonal antibodies, diagnosticsystems for the detection of the presence of a kaposin protein or aderivative thereof as well as antibodies directed against kaposinprotein or a derivative thereof, methods for detection of the expressionof kaposin protein or a derivative thereof in a biological sample,methods for the detection of antibodies directed against kaposin proteinor a derivative thereof, uses of the monoclonal antibodies providedaccording to the invention and uses of kaposin protein or a derivativethereof, each in diagnostics and therapy.

BACKGROUND OF THE INVENTION

The human herpes virus 8 has been detected in all forms of Kaposi'ssarcoma, in primary effusion lymphomas (PEL), in Castleman's disease, inangiosarcomas, in skin lesions of patients who underwenttransplantations, in plasmacytomas, sarcoidosis as well as in healthycontrol individuals (Chang et al., 1994; Boshoff and Weiss, 1997).Seroepidemiological studies have shown that in northern and centralEurope HHV-8 is substantially restricted to risk groups and that thereare large differences with respect to geography and age. Moreover, thesestudies were able to show that in patients with Kaposi's sarcomas aseroconversion for HHV-8 is detectable months to years prior to thediagnosis of Kaposi's sarcoma and that it is likely that HHV-8 istransferred primarily via sexual contacts. To date, the clinical signsof a primary HHV-8 infection are unknown. Due to the detection of HHV-8in almost 100% of all Kaposi's sarcomas, the correlation of the regionalseroprevalence with the incidence of HHV-8 and the seroconversion priorto the appearance of Kaposi's sarcoma it is believed today that HHV-8 atleast represents a co-factor in the tumorigenesis of KS. Up to now, therole of HHV-8 in the other diseases mentioned above is unclear and thequestion whether HHV-8 is involved in other so far unidentified diseasesis still unanswered.

Taxonomically, on the basis of sequence homologies HHV-8 belongs to thegamma herpes virus sub-family and is closely related to EBV andHerpesvirus saimiri. The HHV-8 genome is 140 kb in size and is flankedby several repetitive sequences having a length of approximately 800 bp(Russo et al., 1996). HHV-8 codes for about 80 proteins, 10 of whichshow homology to cellular gene products (Neipel et al., 1997). Similarto all other herpes viruses, HHV-8 is able to cause a lytic infectionwhich then becomes a latent infection. In the latent phase, at least twoviral transcripts are expressed: a differentially spliced mRNA encodingthe v-cyclin, v-flip and LANA proteins, as well as T0.7, a short RNA 0.7kb in length and of up to now unknown function (Zhong et al., 1996). Theviral transcript T0.7 is the most abundant of the RNAs expressed in thelatent phase and has three open reading frames corresponding to 60, 35,and 47 amino acids.

So far, a HHV-8 infection has been detected by polymerase chain reactionusing HHV-8-specific oligonucleotide primers. This direct method ofdetection has disadvantages in that it (i) requires at lot of effort andcosts (about five to ten times the price of serological detection), (ii)is susceptible to false positive results due to contamination, (iii)detects only acute infections but not earlier ones, and (iv) detectsonly 50% of all (acutely) infected subjects if used on peripheral blood.

The serological detection methods developed so far are based on eitherthe use of HHV-8-infected cell lines or on recombinant viral proteins.Assays detecting the antibodies directed against HHV-8 on HHV-8-positivecell lines by means of immunofluorescence have the disadvantages that(i) their reproducibility is low (i.a. because it is impossible to keepthe culture conditions for the HHV-8 cell lines absolutely constant),(ii) their evaluation may not be performed by machine which makes themunsuitable for a larger number of tests, and (iii) it is in partdifficult to exclude cross-reactions of antibodies against otherviruses. A common problem of all of the assays based on recombinantviral proteins ist their low sensitivity. This problem is based on thefact that antibodies are produced only against particular proteins ofthe virus and that different individuals may in part produce antibodiesagainst different proteins. The viral proteins tested to date for theirutility in serological diagnostics have a sensitivity of only between 30and 80%. FIG. 1 exemplarily shows that antibodies against the minorcapsid protein VP23 can be detected in only 30% of KS patients (FIG. 1).It is highly likely that the use of more than one viral protein isrequired in order to develop more sensitive assays.

Since with respect to its frequency Kaposi's sarcoma is in third placeof the tumors occuring after an organ transplantation and according tocurrent knowledge its occurence is closely associated to HHV-8 infectionit may be expected that in the future organ donors and possibly alsoblood products will be tested for HHV-8 similar to e.g. the obligatorytesting for HIV, hepatitis B and C which is performed today.

In part, the dignity of KS is highly variable. The KS which does notoccur endemically is almost exclusively restricted to theimmunodeficient patient and generally has a malignant course. It istreated by chemotherapy (such as liposomal doxorubicin), surgically orby means of radiotherapy with modest success. Retrospective studiesindicate that the virostatics Foscarnet and Gancyclovir are effective.Larger prospective studies with regard to the effectivity of virostaticson KS have not been published to date. Also missing up to now is auniform treatment schedule for the B cell lymphomas in which HHV-8 hasbeen detected. Chemotherapeutic schedules have been widely used whichwere generally employed in the treatment of Non-Hodgekin lymphomas.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide novelmeans for the diagnostics and therapy of HHV-8 infections and ofdieseases directly or indirectly caused by HHV-8.

According to the invention, this object has been achieved by themonoclonal antibodies characterized in claim 1 as well as by thehybridoma cell lines, diagnostic systems, methods of detection, and usescharacterized in more detail in the dependent claims. Preferredembodiments of the invention are obvious from the dependent claims.

According to the invention, there are provided monoclonal antibodiesspecifically recognizing an epitope of the kaposin protein or aderivative thereof and binding to said epitope. An “epitope” accordingto the invention is intended to mean an antigenic determinant of kaposinpeptide or protein or a derivative thereof which may be recognized by amonoclonal antibody and to which the monoclonal antibody can bind.

The derivative of kaposin peptide comprises an amino acid sequence ofkaposin peptide formed by deletion, substitution, insertion, additionand/or chemical modification of one or more amino acid(s) provided thatthe antibody directed against an epitope of the derivative of kaposinpeptide is useful for the specific detection of a HHV-8 infection.

The monoclonal antibody provided by the invention recognizes an epitopein the cytoplasmic region or the extracellular region of kaposin proteinor a derivative thereof.

The monoclonal antibodies used in the present invention are specificallydirected against an epitope of one of the following peptides:

(a) AIPPLVCLLA; or (SEQ ID NO:13)

(b) QRGPVAFRTRVATG. (SEQ ID NO:23)

A further embodiment also comprises the following sequence QRGPVAFRTRVA,(SEQ ID NO:24) composed of the peptides 15 and 16 of Table 1.

In another embodiment of the invention the monoclonal antibody isdirected against a peptide fragment (a derivative) thereof having alength of at least 5, at least 6, or at least 7 amino acids.

“Kaposin protein” according to the invention is intended to mean aprotein having the amino acid sequence shown in FIG. 2 (SEQ ID NO:1) orpartial sequences of the protein (peptides). Comprised by the inventionare also above mentioned peptides and derivatives thereof having anamino acid sequence formed by deletion, substitution, insertion,addition, and/or chemical modification of one or more amino acid(s)provided that the derivative is useful for the specific detection of aHHV-8 infection and/or provided that a monoclonal antibody may bedirected against the derivative of kaposin peptide, useful fordiagnostics and/or therapy of HHV-8 infections and diseases.

Also, the invention relates to hybridoma cell lines producing amonoclonal antibody as characterized in more detail hereinabove.

According to the invention, also diagnostic systems in the form of a kitare provided. These diagnostic systems are present in two embodiments.

In the first embodiment, the diagnostic system serves for the detectionof a kaposin protein or a derivative thereof, as defined above. Thediagnostic system contains at least a monoclonal antibody, as defined inmore detail above, in a container. Typically, the kit systems comprisethe antibody in labeled or unlabeled forms, reagents to perform thenecessary incubations, and substrates or derivatizing agents which willbe employed depending on the labeling used.

The detection of the presence or the expression, respectively, ofkaposin protein or a derivative thereof in a biological sample may beperformed for example as follows: The sample is contacted with amonoclonal antibody, as described above, under conditions such that theantibody is able to bind to an antigenic component of the sample; then,the proportion of antibody binding to the antigenic component in thesample is determined.

As the sample, for example a body liquid, an intact cell, a cellularextract, or a tissue will be used. The proportion of binding of theantibody may be for example determined by immunocytochemical orimmunohistochemical staining.

In a second embodiment of the invention, the diagnostic system isdesigned to be capable of detecting monoclonal or polyclonal antibodiespresent in a sample, such as a body liquid, an intact cell, a cellularextract, or a tissue. For this purpose, a kaposin peptide or aderivative thereof, as defined above and in claim 1, is provided in anappropriate form in a suitable container, the protein or the derivativethereof being capable of undergoing a immunereaction with the monoclonalor polyclonal antibody to be detected. The sample is contacted with thekaposin peptide or a derivative thereof under conditions suitable toenable binding of the antibody present or presumed to be present in thesample directed against the kaposin peptide or a derivative thereof;furthermore, the proportion of binding of the antibody in the sample tothe kaposin peptide of the derivative thereof is determined.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 is a chromatogram and a plot showing the reactivity of sera ofpatients suffering from Kaposi's sarcoma against recombinant minorcapsid protein VP23.

FIG. 2 shows the amino acid sequence of the first T0.7 RNA ORF(“kaposin”).

FIG. 3 shows the data used in the detection of kaposin expression in theHHV-8+cell line BCBL-1 using kap5C4 mab.

FIG. 4 shows the immunohistological staining of KS tissue with mabsagainst lytic HHV-8 VP23 protein.

Thus, the methods of the present invention essentially compriseincubation of the sample to be tested with monoclonal antibodies, or, inthe reverse the incubation of kaposin peptide thereof with a sample tobe tested which is presumed to contain monoclonal antibodies orpolyclonal antibodies directed against kaposin peptide or a derivativethereof. According to the invention, “incubation” may also refer to anyother means of contacting sample material with antibodies. Many possiblebasic methods of performing such immunoassays to be applied are known.These methods comprise for example RIA, ELISA, precipitation methods,agglutination methods, complement-fixation methods, andimmunofluorescence methods. The monoclonal antibodies or the kaposinpeptide or a derivative thereof may be present in labeled form. Thelabels used in the preparation of labeled forms of the antibodies orproteins comprise groups which are directly detectable, such asradiolabels and fluorochromes, as well as groups such as enzymes whichmust be reacted or derivatized for detection. A radiolabel may bedetected by any known method. An enzyme labeling may be detected by anycalorimetric, spectrophotometric, fluoro-spectrophotometric orgasometric technique known per se. The enzyme is linked to the antibodyvia bridging molecules, such as carbodiimide, periodate, diisocyanate,glutaraldehyde. In these methods, many enzymes known per se may beemployed. Examples are peroxidase, alkaline phosphatase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plus peroxidase, galactose oxidase plus peroxidase, and acidphosphatase. Fluorescent materials which may be employed comprise forexample fluorescein and its derivatives, rhodamine and its derivatives,auramine, luciferin, horse-radish peroxidase, alkaline phosphatase,lysozyme, and glucose-6-phosphate dehydrogenase. The antibodies may bebound to these labels by methods known per se. Examples for couplingagents are aldehydes, carbodiimides, dimaleimides, imidates,succinimides, to provide the antibodies with the above-describedfluorescent, chemiluminescent and enzyme labels.

The antibodies and labeled antibodies and the peptides produced here maybe used to detect diseases directly or indirectly caused by HHV-8, suchas B cell lymphomas, in a patient and/or to monitor the status of thedisease. For this purpose either qualitative or quantitative immunoassaymethods may be employed. The assay techniques comprise direct andindirect detection procedures. If the sample contains for exampleKaposi's sarcoma cells, the labeled antibody will bind to these cells.Following washing of the tissue or the cells to remove unbound labeledantibody the sample is examined for labeled immune complexes. In theindirect detection methods, the tissue or the cell sample or a bodyliquid is incubated with unlabeled monoclonal antibody. Then, the sampleis treated with a labeled antibody directed against the monoclonalantibody, washed, and the presence of ternary complexes is examined.

For diagnostic purposes, the antibodies and also the kaposin peptideswill be typically present in kit form.

Description of Therapeutical uses of the Antibodies

In an in vivo therapy the present antibodies are administered intherapeutically effective amounts (i.e. amounts abolishing oralleviating the disease directly or indirectly caused by HHV-8) to apatient. This is generally done by the parenteral or intravenous route.The dose and the formulation are dependent on the nature of the diseaseto be treated and the patient.

The derivative of the kaposin peptide employed according to theinvention or the derivative of the kaposin peptide to be detectedaccording to the invention preferably comprises an amino acid sequenceof the kaposin peptide formed by deletion, substitution, insertion,addition and/or chemical modification of one or more amino cid(s).However, the final product of a derivatization process of that type isalways designed to enable a specific detection of a HHV-8 infection or adisease induced by HHV-8 infection.

In this invention, the kaposin peptide or its derivative each comprisesan epitope in the cytoplasmic region and the extracellular region ofkaposin protein or a derivative thereof. The peptides have the followingamino acid sequence:

(a) AIPPLVCLLA; or (SEQ ID NO:13)

(b) QRGPVAFRTRVATG. (SEQ ID NO:23)

A further embodiment shows the following sequence:

QRGPVAFRTRVA. (SEQ ID NO:24)

The fragments of the kaposin peptides have a length of at least 5, atleast 6 or at least 7 amino acids. The monoclonal antibodies providedaccording to the invention are directed against kaposin peptide or thederivatives of the kaposin peptides described in more detail above.

The present antibodies are employed in diagnostics and therapy ofdiseases caused directly or indirectly by infections with HHV-8. Theseinclude for example Kaposi's sarcoma, AIDS diseases and B celllymphomas.

Also, the kaposin peptides provided by the invention or the derivativesof kaposin peptides characterized in more detail above are used indiagnostics and therapy of HHV-8 infections, AIDS diseases, Kaposi'ssarcomas and B cell lymphomas.

In the following, the invention will be described in more detail withrespect to Examples and the accompanying Figures. The Figures show:

FIG. 1: Reactivity of sera of patients suffering from Kaposi's sarcomaagainst recombinant minor capsid protein VP23

(a) The minor capsid protein VP23 of HHV-8 was expressed as GST-taggedprotein in E. coli, purified via affinity chromatography by glutathionesepharose and employed in an ELISA for the detection of anti-VP23antibodies in patient sera. (b) Microtiter plates were coated with 1 μgof recombinant VP23 and blocked by PBS/0.1% Tween 20. The sera of 19HIV-negative control donors, 19 HIV-positive individuals without KS and36 HIV-negative patients with KS were pre-incubated with lyophilizedE.coli and then incubated in a dilution of 1:50 for 2 hours at roomtemperature on the microtiter plate. After 4 washing steps, aperoxidase-conjugated secondary antibody and afterwards ABTS substratewere added. The sera of the HIV-positive KS patients showed a clearreactivity against VP23 (Wilcoxon test p<0.0024) but only about ⅓ of thesera was higher than a cut-off of OD 0.2. Therefore, the assay may behardly used for the detection of HHV-8 infection.

FIG. 2: Amino Acid Sequence of the First T0.7 RNA ORF (“kaposin”)

In the kaposin amino acid sequence (SEQ ID NO:1), the presumabletransmembrane region (SEQ ID NO:3)(underlayed by dark box), the bindingsite of kap5C4 mab (SEQ ID NO:24)(underlayed by hatched box) and asecond hydrophobic region (underlayed by light box) with so far unknownfunction have been indicated. The peptides used for epitope mapping (seealso Table 1(SEQ ID NOS:5-22)) are shown in the lower panel of theFigure. The cytoplasmic region (SEQ ID NO:2) and extracellular region(SEQ ID NO:4) are also indicated.

FIG. 3: Detection of Kaposin Expression in the HHV-8+ Cell Line BCBL-1Using kap5C4 mab

(a) Detection of kaposin by Western Blot in uninduced HHV-8+ BCBL-1cells but not in the EBV+ control cell line 721. (b) Detection ofkaposin in fixed/permeabilized (upper panel) and unpermeabilized (lowerpanel) BCBL-1 cells by immunofluorescence and evaluation using aconfocal laser microscope. In both of the left panels the cells shown byimmunofluorescence on the right are shown by means of phase contrastphotography. The uninduced BCBL-1 cells were first reacted withundiluted cell culture supernatant of kap5C4 hybridoma and then with aCy-3-labeled secondary antibody. (c) Evaluation of the kap5C4-stainedcells shown above in the flow cytometer (bold curve). As the positivecontrol, an antibody W6/32 directed against MHC class 1 was used (dottedcurve). In the negative control, incubation was performed only with thesecondary antibody (plain curve).

FIG. 4: Immunohistological Staining of KS Tissue with Mabs Against LyticHHV-8 VP23 Protein

The formaline-fixed and paraffin-embedded thin section of a Kaposi'ssarcoma was stained with vp4G2 mab against HHV-8 VP23. First, thesection was cleared from paraffin by boiling and afterwards incubatedwith a 1:10 dilution of vp4G2 mab. Bound antibodies were detected usingthe EnVision™ system (Dako, Hamburg).

Cells infected by HHV-8 in a latent manner express at least 2 viraltranscripts. The most abundant viral mRNA in the latent phase, T0.7, hasa length of 0.7 kb and three short open reading frames (ORFs). Up tonow, no translation of any of these ORFs has been demonstrated. Thefirst ORF has a length corresponding to only 60 amino acids. In the bulkof the literature only two transmembrane proteins are described whichare smaller than 14 kd (ponticulin and L-selectin), and this is thereason why it was unclear whether T0.7 is translated at all or if itlike the T1.1/nut-1 transcript (equally encoding an ORF of 62 aminoacids in length) discovered at the same time has a regulatory function.The patent application is based on the following studies of the firstORF of T0.7, called “kaposin”:

1. Expression of the First ORF of T0.7 Transcript (“kaposin”) inHHV-8-infected Cells

The applicant was able to show that the first open reading frame of theT0.7 transcript, “kaposin”, is indeed translated in HHV-8-infectedcells. The sequence of the first ORF was expressed in E. coli in theform of a GST (glutathione-S-transferase) fusion protein and purifiedusing affinity chromatography. The recombinant fusion protein was usedfor the preparation of monoclonal antibodies (mab). For this purpose,Lou/C rats were immunized and the spleen cells were fused with themyeloma cell line P3C63Ag8.653 according to standard techniques. Theantibodies were screened with recombinant fusion protein by ELISA andrescreened with recombinant GST. The kap5C4 mab recognizes recombinantkaposin fusion protein but not GST in the Western blot. By means ofpeptide mapping, kap5C4 mab was found to recognize the peptide sequenceQRGPVAFRTRVA (SEQ ID NO:24) at the C-terminus (FIG. 2 and Tab. 1). Incell lysates of HHV-8-infected cell lines a protein could be identifiedby Western blot, immunoprecipitation and following surface biotinylationhaving a length of 6 kd predicted on the basis of the sequence (FIG.3a).

2. Expression of Kaposin on the Cell Surface

It was shown by immunofluorescence that kaposin is expressed on thesurface of HHV-8-infected cell lines (FIGS. 3b and c). The FIG. 3b showsthat kaposin is recognized by kap5C4 mab in fixed and permeabilizedBCBL-1 cells (HHV-8-positive, EBV-negative cell line established from aprimary effusion lymphoma). Further, the Figure shows that kaposin isalso detectable on unpermeabilized cells and therefore must be localizedon the cell surface. The immunofluorescence shows staining on theperiphery typical for cell surface molecules. The FIG. 3c shows aflow-cytometric analysis of BCBL-1 cells stained with kap5C4 or acontrol antibody against MHC class 1. It is obvious that the expressionwith kap5C4 mab ist clearly positive about one log higher than thenegative control. Since kap5C4 recognizes the C-terminus of kaposin theresults must be interpreted in a way that kaposin is a type II protein(N-terminus is localized in the cytoplasm).

3. Expression of Kaposin in the Majority of HHV-8-Infected Cells

Lytic proteins such as the VP23 minor capsid protein studied by theapplicant are expressed by HHV-8+ cell lines only after induction withphorbol esters and/or n-butyrate. By immunohistology, in most of the KStissues only a very small portion of spindle cells is positive withvp4G2 mab against VP23 (FIG. 4). In contrast, HHV-8+ cell lines expresskaposin in a constitutive manner without previous induction. Dependingon the cell line and staining method the percentage of positive cellsvaries between 10 and more than 80%. Also by immunohistology, the numberof cells stained by kap5C4 mab is much greater than the vp4G2-positivecells. Presumably, with a more sensitive staining method a major portionof all spindle cells would be positive for kaposin.

4. Recognition of Kaposin by Sera of KS Patients and KS Risk Groups

It was shown by Western blot that kaposin fusion protein is recognizedby sera of KS patients and of HIV-infected individuals without KS,respectively. It was demonstrated that a higher percentage (approx. 80%)of the sera of the HIV-infected (risk group: homosexuals) reacted withkaposin as compared to other viral proteins/peptides. This may indicatethat kaposin as a latent viral membrane protein is more suitable for thedetection of early infections than lytic proteins. By peptide-ELISA arough mapping was performed to determine the epitope recognized by thesesera. It was demonstrated that a hydrophobic peptide having the aminoacid sequence AIPPLVCLLA (SEQ ID NO:13) was recognized by most of thesera (Tab. 1).

Advantages Over the Prior Art and Potential Utilization

As outlined above, the detection methods used to date of a HHV-8infection suffer from many disadvantages. At the moment no therapyspecifically directed against HHV-8 exists. The viral protein kaposindescribed herein has the following advantages over the viral proteinsused so far:

1. Expression on the cell surface

2. Expression in a majority of HHV-8-infected cells

3. Kaposin is the latent HHV-8 protein showing the best expression

4. No homologies to other proteins (also viral) are known ot date

The potential uses become obvious from the function of kaposin as areliable infection or tumor marker which according to present data iswell expressed by a majority of HHV-8-infected cells. Therefore, kaposinis useful for the development of (i) novel diagnostic methods for thedetection of HHV-8 infection and (ii) of therapeutics specificallydirected against HHV-8. In the field of diagnostics for example the useof recombinant kaposin in ELISAs or Western blots for serologicaldetection or the use of mab against kaposin for the detection ofHHV-8-infected cells in immunohistology may be contemplated. A reliableserological assay has the following possibilities of application:

Diagnosis of HHV-8 infection;

prognostic marker for the development of a KS sarcoma in HIV-infectedindividuals, patients after transplantation and immunosuppressedpatients;

course parameter in the therapy of Kaposi's sarcoma;

diagnosis of certain B cell lymphomas;

course parameter in the therapy of HHV-8+ B cell lymphomas;

possibly in the diagnosis of so far unknown tumors (e.g. also so-called“secondary tumors” following organ transplantations) and possibly otherdiseases so far not connected with HHV-8;

course parameter in these HHV-8-associated diseases.

In principle, also a therapeutic utilization of kaposin may becontemplated, e.g. if bispecific antibodies are used which bycross-linking of HHV-8+ tumor cells with T lymphocytes may lead totargeted activation of the immune response and thus to an elimination ofthe tumor cells. Since up to now the mechanism by which HHV-8-infectedcells can be transformed is unknown, kaposin as a latent protein may beinvolved as the causative agent. If this should prove to be true atherapeutical intervention leading to a blockage of kaposin may be oftherapeutical benefit.

Cell Lines

The cell lines BCBL-1 (HHV-8+, EBV-) (kindly provided by Don Ganem;UCSF, USA) and 721 (EBV+ lymphoblastoid cell line) used were passaged inRPMI (Life Technologies, Paisley, UK) supplemented with 20% ofheat-inactivated fetal calf serum, 100 IU/ml of penicillin, 100 μg/ml ofstreptomycin, 2 mM L-glutamine, and 0.05 mM 2-mercaptoethanol (Sigma,St. Louis, USA).

Preparation of Recombinant HHV-8 Fusion Proteins

The sequence of the first ORF of T0.7 was amplified from extracted DNAof BCBL-1 cells by PCR, cloned into pGEX-4T vector (Pharmacia, Uppsala,Sweden), expressed in E. coli as a GST (glutathione-S-transferase)fusion protein and purified by affinity chromatography according tostandard techniques (Ausubel et al., 1992).

Preparation of Monoclonal Antibodies Against Kaposin

The monoclonal antibodies were prepared according to standard methods asdescribed previously (Kremmer et al., 1995). Lou/C rats were immunizedin intervals of 3 weeks with the recombinant fusion protein andtrehalose dimycolate/monophosphoryl lipid A/squalene/Tween 80 (AntibodyMultiplier ABM-S, Linaris, Bettingen, Germany) as the adjuvant for atotal of three times. Spleen cells of the immunized rats were fused withP3C63Ag8.653 myeloma cell line and the hybridoma supernatants weretested by means of ELISA (enzyme-linked immunosorbent assay) onmikrotiter plates coated with the recombinant protein in a concentrationof 1 μg/well. The supernatants were incubated for 1 hour and the boundantibodies detected using a peroxidase-conjugated secondary antibodyagainst rat immunoglobulin. Afterwards, o-phenylene diamine was added asthe substrate and the absorbance measured in an ELISA reader (Tecan,Reading, UK). In total, antibody kap5C4 was cloned twice.

Western Blotting

Cell lysates of unstimulated BCBL-1 and 721 cells were separated on a18% SDS PAGE and blotted onto nitrocellulose (Schleicher and Schuell,Dassel, Germany). Afterwards, the blot was blocked with 5% milk powder(Merck, Darmstadt, Germany) in TBS (tris-balanced saline)/0.02% Tween-20and incubated with kap5C4 mab. Following three washing steps inTBS/0.02% Tween-20 the blot was incubated for one hour with alkalinephosphatase-conjugated secondary antibody (Dianova, Hamburg, Germany) ina dilution of 1:1000, again washed and finally incubated with WesternBlue (Promega, Madison, USA) according to the manufacturer'sinstructions.

Immunofluorescence

For intracellular staining, the uninduced BCBL-1 cells were fixed inaceton and permeabilized with 0.2% saponine. Untreated BCBL-1 cells wereused for surface staining. The BCBL-1 cells were first incubated withundiluted kap5C4 hybridoma cell culture supernatant and then with aCy-3-labeled secondary antibody (Sigma, St. Louis, USA). For analysis,the cells were fixed on poly-L-lysine-coated slides (Marienfeld, BadMergentheim, Germany) and analyzed by Leica TNT confocal lasermicroscope (Leica, Bensheim, Germany). For control in flow cytometryW6/32 mab against MHC class I was used as the primary antibody. Analysiswas conducted on a FACSscalibur flow cytometer (Becton Dickinson,Mountain View, USA).

Elisa

The microtiter plates were coated with 1 μg of recombinant VP23 andblocked by PBS/0.1% Tween-20. Sera of 19 HIV-negative control donors, 19HIV-positive individuals without KS and 36 HIV-negative patients with KSwere pre-incubated with lyophilized E. coli and afterwards incubated ina dilution of 1:50 for two hours at room temperature on the microtiterplate. After 4 washing steps a secondary peroxidase-conjugated antibodyfollowed by ABTS substrate (Boehringer, Mannheim, Germany) was added.

Peptides were used to perform the peptide ELISA which were linked attheir N termini to a SGSG spacer and were biotinylated. A stock solutionof each of the peptides (concentration of 1 μmole/ml in 100% DMSO) wasdiluted 1:400 in PBS/5% Tween-20 and bound to streptavidin-coatedmicrotiter plates. Following an incubation with a dilution of 1:200 ofkap5C4 mab hybridoma supernatant and 1:50 of KS patient serum,respectively, a peroxidase-conjugated secondary antibody against rat IgG(kap5C4) or human IgG was used in a dilution of 1:10.000. After reactionwith the secondary antibody the microtiter plates were incubated withABTS substrate and then measured with an ELISA reader at a wavelength of492 nm. To identify epitopes against which the antibodies inHHV-8-infected persons are directed a serum pool of HIV-positivepatients (<10) suffering from Kaposi's sarcoma was used.

Immunohistology

First, the section was cleared of paraffin and then incubated with vp4G2mab in a dilution of 1:10. Bound antibodies were detected using theEnVision™ system (Dako, Hamburg, Germany).

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TABLE 1 Epitope mapping of kap5C4 monoclonal antibody and of KS patientsPeptide Sequence Reactivity Reactivity KS SEQ Name (N→C) kap5C4 mabpatient sera ID NO: 1. K12A MDRGLTVFVA − −  5 2. K12A2 LTVFVAVHVP − −  63. K12A3 VAVHVPDVLL − −  7 4. K12A4 HVPDVLLNGW − −  8 5. K12A5DVLLNGWRWR − −  9 6. K12A6 LNGWRWRLGA − − 10 7. K12B WRWRLGAIPP − − 118. K12C RLGAIPPLVC − − 12 9. K12D AIPPLVCLLA − +++ 13 10. K12D2PLVCLLAISV − − 14 11. K12D3 CLLAISVVPP − − 15 12. K12D4 AISVVPPSGQ − −16 13. K12D5 VVPPSGQRGP − − 17 14. K12D6 PSGQRGPVAF − − 18 15. K12EQRGPVAFRTR +++ − 19 16. K12F GPVAFRTRVA +++ − 20 17. K12G VAFRTRVATG − −21 18. KI2H FRTRVATGAH − − 22

The peptides indicated are linked at their N-terminus to a SGSG (SEQ IDNO:25) spacer and biotinylated. A stock solution of each of the peptides(concentration of 1 μmole/ml in 100% DMSO) was diluted 1:400 in PBS/5%Tween-20 and bound to streptavidin-coated microtiter plates. Followingan incubation with a dilution of 1:200 of kap5C4 mab hybridomasupernatant and 1:50 of KS patient serum, respectively, aperoxidase-conjugated secondary antibody against rat IgG (kap5C4) orhuman IgG was used in a dilution of 1:10.000. After reaction with thesecondary antibody the microtiter plates were incubated with ABTSsubstrate and then measured with an ELISA reader at a wavelength of 492nm. To identify epitopes against which the antibodies in HHV-8-infectedpersons are directed a serum pool of HIV-positive patients (<10)suffering from Kaposi's sarcoma was used.

25 1 60 PRT human herpesvirus 8 kaposin - first T0.7 RNA ORF 1 Met AspArg Gly Leu Thr Val Phe Val Ala Val His Val Pro Asp Val 1 5 10 15 LeuLeu Asn Gly Trp Arg Trp Arg Leu Gly Ala Ile Pro Pro Leu Val 20 25 30 CysLeu Leu Ala Ile Ser Val Val Pro Pro Ser Gly Gln Arg Gly Pro 35 40 45 ValAla Phe Arg Thr Arg Val Ala Thr Gly Ala His 50 55 60 2 26 PRT humanherpesvirus 8 DOMAIN (1)..(26) cytoplasmic region 2 Met Asp Arg Gly LeuThr Val Phe Val Ala Val His Val Pro Asp Val 1 5 10 15 Leu Leu Asn GlyTrp Arg Trp Arg Leu Gly 20 25 3 16 PRT human herpesvirus 8 TRANSMEM(1)..(16) putative transmembrane region 3 Ala Ile Pro Pro Leu Val CysLeu Leu Ala Ile Ser Val Val Pro Pro 1 5 10 15 4 18 PRT human herpesvirus8 DOMAIN (1)..(18) extracellular region 4 Ser Gly Gln Arg Gly Pro ValAla Phe Arg Thr Arg Val Ala Thr Gly 1 5 10 15 Ala His 5 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12A 5 Met Asp Arg Gly Leu Thr Val PheVal Ala 1 5 10 6 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12A2 6Leu Thr Val Phe Val Ala Val His Val Pro 1 5 10 7 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12A3 7 Val Ala Val His Val Pro Asp ValLeu Leu 1 5 10 8 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12A4 8His Val Pro Asp Val Leu Leu Asn Gly Trp 1 5 10 9 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12A5 9 Asp Val Leu Leu Asn Gly Trp ArgTrp Arg 1 5 10 10 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12A6 10Leu Asn Gly Trp Arg Trp Arg Leu Gly Ala 1 5 10 11 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12B 11 Trp Arg Trp Arg Leu Gly Ala IlePro Pro 1 5 10 12 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12C 12Arg Leu Gly Ala Ile Pro Pro Leu Val Cys 1 5 10 13 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12D 13 Ala Ile Pro Pro Leu Val Cys LeuLeu Ala 1 5 10 14 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12D2 14Pro Leu Val Cys Leu Leu Ala Ile Ser Val 1 5 10 15 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12D3 15 Cys Leu Leu Ala Ile Ser Val ValPro Pro 1 5 10 16 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12D4 16Ala Ile Ser Val Val Pro Pro Ser Gly Gln 1 5 10 17 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12D5 17 Val Val Pro Pro Ser Gly Gln ArgGly Pro 1 5 10 18 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12D6 18Pro Ser Gly Gln Arg Gly Pro Val Ala Phe 1 5 10 19 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12E 19 Gln Arg Gly Pro Val Ala Phe ArgThr Arg 1 5 10 20 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12F 20Gly Pro Val Ala Phe Arg Thr Arg Val Ala 1 5 10 21 10 PRT humanherpesvirus 8 PEPTIDE (1)..(10) K12G 21 Val Ala Phe Arg Thr Arg Val AlaThr Gly 1 5 10 22 10 PRT human herpesvirus 8 PEPTIDE (1)..(10) K12H 22Phe Arg Thr Arg Val Ala Thr Gly Ala His 1 5 10 23 14 PRT humanherpesvirus 8 23 Gln Arg Gly Pro Val Ala Phe Arg Thr Arg Val Ala Thr Gly1 5 10 24 12 PRT human herpesvirus 8 24 Gln Arg Gly Pro Val Ala Phe ArgThr Arg Val Ala 1 5 10 25 4 PRT Artificial Sequence Description ofArtificial Sequencespacer 25 Ser Gly Ser Gly

What is claimed is:
 1. Monoclonal antibody wherein said antibodyspecifically recognizes and binds an epitope selected from the groupconsisting of: (a) AIPPLVCLLA (SEQ ID NO:13), (b) QRGPVAFRTRVATG (SEQ IDNO:23), and (c) derivatives of (a) and (b) that contain at least 5contiguous amino acids and that are formed by deletion, substitution,insertion, addition, and/or chemical modification of an amino acid ofpeptides (a) or (b), with the proviso that said antibody is able tospecifically detect a HHV-8 infection.
 2. Diagnostic system in the formof a kit comprising a monoclonal antibody according claim 1 in acontainer, said antibody being for the detection of the presence of akaposin protein having an amino acid sequence selected from the groupconsisting of AIPPLVCLLA (SEQ ID NO:13), ORGPVAFRTRVATG(SEQ ID NO:23),and derivatives of AIPPLVCLLA and ORGPVAFRTRVATG that are formed bydeletion, substitution, insertion, addition, and/or chemicalmodification of an amino acid of sequences AIPPLVCLLA or ORGPVAFRTRVATG,with the proviso that said antibody is able to specifically detect aHHV-8 infection.
 3. Method for the detection of the presence of kaposinprotein or a derivative thereof in a biological sample wherein saidsample is contacted with a monoclonal antibody according to claim 1under conditions such that said antibody is able to bind to an antigeniccomponent in the sample and binding of the antibody to the antigeniccomponent in the sample is determined.
 4. Method according to claim 3wherein said sample is a liquid, an intact cell, a cellular extract or atissue.
 5. Method according to claim 3 wherein the proportion of bindingof said antibody is determined by immunocytochemical orimmunohistochemical staining.